The trace metal copper is an essential nutrient which is also a potent toxin when allowed to accumulate to high free intracellular levels. This dilemma requires that cells harbor finely tuned mechanisms for the regulation of copper uptake, distribution, sequestration and efflux to establish and maintain copper homeostasis. The existence of at least two human genetic disorders of copper homeostasis, which are well characterized clinically yet undefined with respect to the nature of the defect, underscores the importance of copper homeostasis. The experiments proposed in this application focus on understanding several aspects of copper homeostasis in two eukaryotic microorganisms, the baker's yeast Saccharomyces cerevisiae and the opportunistic yeast Candida glabata. The first specific aim of this proposal is to investigate the mechanisms by which the S cerevisiae CUP1 gene, encoding a highly efficient copper binding protein called metallothionein (MT), is transcriptionally regulated. The mode of action of previously identified CUP1 transcription factors including ACE1, HSF and ACE2, and the mechanisms underlying CUP1 regulation in response to carbon source and oxygen, will be investigated using genetic, biochemical and molecular approaches. Secondly, detailed molecular and genetic studies of copper-activated MT gene transcription will be carried out in C glabrata, a yeast which exhibits the MT gene organization typical of higher eukaryotes, but the metal specificity for MT gene induction similar to that of S cerevisiae. Third, genetic and molecular approaches will be used to isolate other genes form S cerevisiae which participate in the process of copper homeostasis. The genes and gene products will be characterized and cells bearing deletions of these genes will be studied to ascertain the role these genes play in copper homeostasis. These studies should contribute a great deal toward the long range goal of understanding the mechanisms for copper homeostasis in biological systems.